Method for producing hyaluronidase conjugate with polyethylenepiperazine derivatives and the use of the conjugate produced

ABSTRACT

This invention relates to a method for obtaining immobilized enzyme preparations, in particular to preparation and application of noval active conjugate of enzyme with a polymer carrier. Said conjugate possesses the properties of Longidaza drug and inhibits hyperplasia of connective tissue, and anti-inflammatory action, as well as it can be used for manufacturing stable, active and safe in use long-acting drugs in the form of a suppository, ointment, injection or cosmetic cream, and for making veterinary drugs. This method consists in conjugation of hyaluronidase with a water-soluble copolymer using carbodiimide or azide method. The conjugation is carried out with the use of copolymer N-oxide 1,4-ethylene piperazine, (N-carboxymethyl)-1,4-ethylene piperazine or its hydrazide, and 1,4-ethylene piperazine of general formula 
     
       
         
         
             
             
         
       
         
         where n is ranging from 40% to 90% of total number of cells; 
         m is ranging from 3% to 40% of total number of cells; 
         n+m+1=100%,

This invention relates to development of technologies for obtainingimmobilized enzyme preparations. It can be widely used in variousbranches of industry, particularly in pharmaceutical industry, forproducing stable, active and safe-in-application long-acting medicinalpreparations (drugs).

The most effective method of stabilization of compounds of protein orother nature in a physiological solution consists in chemicalconjugation based on high-molecular carriers [Parveen S, Sahoo S. K.,Clin Pharmacokinet. 2006; 45(10), p. 965; Duncan R., PEGylated ProteinDrugs: Basic Science and Clinical Applications. Ed.: Birkhäuser Basel2009; Harris J. M, Chess R B. Effect of pegylation on pharmaceuticals.//Nat Rev Drug Discov, 2003, 2(3), p. 214.

Covalent binding of medicinal proteins with a polymer carrier increasesmanifold conformational stability of protein molecules and theirresistance to protease and specific inhibitors effect, which makespossible creation of micromolecular bioactive long-acting drugs based onthem [Nekrasov A. V., Puchkova N G, Immunology (Immunologiya), 2006;27(2), p. 1].

According to present knowledge, there are plenty polymer carriers usedto prepare conjugates with protein and non-protein compounds. The mostknown are the conjugates based on polyethylene glycole (PEG), used asmedicinal preparations [Beilon P. S., Palleroni A. B./InterferonConjugates (Conjugaty interferona), Patent RU 2180595. Burg Y, HilgerB., Josel H.-P., Patent RU 2232163; Kurochkin S. N., Parkansky A. A.,Patent RU 2298560].

There are known water-soluble polymer heterocyclic amines, for example,derivatives of N-oxide poly-1,4-ethylene piperazin. Polymer amines areunique in their properties and applicability as a polymer carrier forconjugation. Copolymer of N-oxide 1,4-ethylene piperazin and(N-carboxymethyl)-1,4-ethylenepiperazin halogenide (Polyoxidonium) isnon-toxic. It possesses antiradical and detoxification properties, andit is bio-degradable due to N-oxide groups. This copolymer is allowedfor application in medical practice, and it is used as animmune-modulator, adjuvant or polymer carrier [Nekrasov A. V., PuchkovaN. G, Ivanova A. S. Derivatives of poly-1,4-ethylene piperazin havingimmune-modilating, antivirus, antibacterial activities. Patent RU2073031]. This makes urgent and important the directions of developmentof easy-to-reach and simple technologies of production of polymercarriers, compliance with all specified requirements, and variousconjugation processes on their basis.

Patent RU 2185388 (published on 20 Jul. 2002) describes oxidation ofpoly-1,4-ethylene piperazin in an aqueous solution containing a solventand an oxidizer and being capable to produce atomical oxygen undernormal conditions. It is appropriate to use as an oxidizer organic andinorganic peroxides and hydroperoxides, salts of oxygenated haloacids,ozone, oxygen, all resulting from water electrolysis.

There can be used as an acidic vehicle, for example, strong acetic acidwater solution.

It is reasonable to perform oxidation by way of mixing poly-1,4-ethylenepiperazin (PEP) with acetic acid (AA) aqueous solution and hydrogenperoxide (HP) in a molecular ratio: PEP:AA:HP=1:0.45:0.7. The oxidationprocess goes on in a heterogeneous medium until complete dissolution ofthe polymer is reached. Then the obtained N-oxide poly-1,4-ethylenepiperazin is subjected to alkylation in the presence of an alkylatingagent. This process is carried out in aqueous environment. It isrecommended to use as an alkylating agent the substances coming incovalent bonding with a tertiary nitrogen atom in a polymer chain withina temperature range from 30 to 100° C., for example, haloacids or theirethers of cyclic or acyclic construction.

It is preferable to use bromo-acetic acid ester as the alkylating agent.

The solution of copolymer of N-oxide poly-1,4-ethylene piperazin and(N-carboxyethyl)-1,4-ethylene piperazin bromide (C—PNO) obtained in theprocess of alkylation, upon its cleaning, for example, byultrafiltration, can be used for further synthesis of high-molecularwater-soluble biogenic compounds, particularly conjugates or complexesof biologically active substances.

Patent RU 2556378 (published on 10 Jul. 2015) describes the conjugate ofglycoprotein having activity of erythropoietin and a method of itsproduction. Said method includes oxidation of polyethylene piperazin toN-oxide, and alkylation by bromo-acetic acid with resultant copolymerN-oxide poly-1,4-ethylene piperazin and (N-carboxymethyl)-1,4-ethylenepiperazin bromide, and cleaning of obtained copolymer. Copolymerconjugation with erythropoietin is carried out by carbodiimide orhydrazide method.

The closest to the claimed invention is the invention described inPatent RU 2112542 (published on 10 Jun. 1998). This Patent relates tothe drug comprising conjugate of hyaluronidase enzyme for treatment ofpathological state of connective tissues. This Patent describes a methodfor obtaining a conjugate of hyaluronidase enzyme with high-molecularcarrier—copolymer of N-oxide poly-1,4-ethylene piperazin and(N-carboxyethyl)-1,4-ethylene piperazin bromide (Polyoxidonium), mol.wt. 40,000-100,000 D, and enzyme:carrier ratio of 1:(1-5) correspondingto the general formula

where R is the hyaluronidase enzyme; n=300-700 is the number of unitunits; q=0.2-0.4 is the number of alkylated units; z=0.4-0.8 is thenumber of oxygenated units. At that, hyaluronidase, segregated frombovine seminal glands, can be used as enzyme in the drug. This methodconsists in conjugation of hyaluronidase with Polyoxidonium using eitheran azide method or a method of activated succinimide ethers. When usingazide method the conjugate is obtained in two steps: at the first stepPolyoxidonium hydrazide is obtained from Polyoxidonium, and at thesecond step the conjugate is obtained by way of reaction ofPolyoxidonium azide coupling with an enzyme. When using the method ofactivated ethers, first, a Polyoxidonium succinimide ether is obtained,and then it is conjugated with an enzyme. The conjugated drug was namedas Longidaza. The method for obtaining copolymer of N-oxidepoly-1,4-ethylene piperazin and (N-carboxyethyl)-1,4-ethylene piperazinbromide is not described in this Patent.

Great demand for preparations having the properties of Longidaza drug atthe pharmaceutical market, economic and environment requirementsprompted improvement of the process used for production with a purposeof ruling out some disadvantages and gaining rise in productivity.

All above-mentioned Patents do not describe distribution of molecularunits in the copolymer structure, or the conjugate obtained on itsbasis.

The studies revealed that distribution of molecular units in thecopolymer structure exerts effect upon the properties of the conjugateproduced, namely, on the degree of conjugation, outputs of targetproduct and stability of conjugated preparations.

The present invention object consists in development of a method forgetting a conjugate of hyaluronidase enzyme with a polymer carriercomprising the copolymer of N-oxide poly-1,4-ethylene piperazin and(N-carboxymethyl)-1,4-ethylene piperazin bromide having at the same timethe properties of inhibition of hyperplasia of connective tissue andanti-inflammatory action, being fit for treatment of pathological stateof connective tissues with improved properties and increased methodoutput.

The assigned task can be solved by a method for obtaining activeconjugate of hyaluronidase enzyme with a copolymer comprising N-oxide1,4-ethylene piperazin and (N-carboxymethyl)-1,4-ethylene piperazinhalogenide using carbodiimide or azide method for conjugation, cleaningand spray-freeze drying. The conjugation is performed with the use ofwater-soluble copolymer, which is a copolymer of N-oxide 1,4-ethylenepiperazin, (N-carboxymethyl)-1,4-ethylene piperazin or its hydrazidederivative and 1,4-ethylene piperazin of general formula (I)

where n is ranging from 40% to 90% of total number of units;m is ranging from 3% to 40% of total number of units;n+m+1=100%,

obtained from poly-1,4-ethylene piperazin with the use of the step ofoxidation by means of an oxidizing agent being able to generate atomicaloxygen under normal conditions in the presence of urea, alkylation bymeans of lower haloalkane acid or its alkyl ether, and hydrazinolysis inthe case of azide method.

Urea is added at the oxidation step in amount of 1 to 10 wt. %,preferably 3 to 6%, per total mass of reaction feed, water included.

The conjugation is carried out with the use of hyaluronidase taken frombovine seminal glands.

Cleaning is carried out by washing with purified water onsemitransparent cassette with the lower limit of particles cutoffranging from 1 to 30 kDa.

The copolymer used is a random polymer, the units of which in themolecule structure are in disordered state and can be in any order andcombination.

The profile of distribution of functional groups influences the polymercarrier reactivity in conjugation reaction, as well as stability ofcopolymer and conjugates based on this copolymer.

The copolymer derived by using various methods and having the samequantity with similar active groups (N-oxide and carboxyl groups)demonstrates absolutely different reactivity in conjugation reaction.This is due to that carboxyl groups can be difficult to access becauseof steric reasons and/or not be activated when N-oxide groups arebehind. Usage of a polymer carrier with low-reactive carboxyl groupsleads to significant decrease of conjugation yield and extent, worsensmolecular weight distribution of derived conjugates. Up to the presentinvention the heterocyclic N-oxide polymer carrier did not have therequired number of reactive groups (carboxyl or hydrazide ones), whichmade impossible getting reliably high-degree of conjugation byhyaluronidase with enzyme using carbodiimide or hydrazide conjugationmethod. The methods of production claimed in the present inventionprovide for getting hyaluronidase preparations having the degree ofconjugation of not less than 90% even on a wholesale scale.

Availability of big portions of copolymer molecule comprising onlyN-oxide groups makes polymer molecule fragile (sensitive?). Thestability study demonstrated that conjugates based on such polymermolecules with time undergo degradation. The rate of degradation of apolymer carrier with irregular distribution of N-oxide groups is notconstant, the degradation occurs rather quickly during the first monthsof storage. On the contrary, the polymer carrier, having uniformdistribution of N-oxide groups, is much more stable, its degradationrate is less and constant. The conjugate stability and uniformity ofN-oxide groups distribution in the polymer carrier also depends on theconditions of the method for copolymer production for conjugation (seeTable 1 and Table 2).

The claimed invention relating to the method for production ofhyaluronidase conjugates provides for production of a polymer carrierhaving not only preset quantitative data (quantity of various units in apolymer), but the required distribution of units, which qualitativelychanges the properties of both the polymer carrier proper, and aconjugate with an enzyme on its base.

The invention makes it possible to produce safe, high-efficiency andstable medicinal preparation (drug) possessing the properties ofsimultaneously inhibiting connective tissue hyperplasia andanti-inflammatory action, and representing a conjugate of hyaluronidase(a therapeutic enzyme taken from bovine seminal glands) andwater-soluble copolymer of general formula (I).

In the case of azide method of conjugation alkylation is carried outwith the use of alkyl ether of haloalkane acid, and alkylation step andhydrazinolysis are combined. The conjugation with hyaluronidase isperformed by azide method at temperatures ranging from 0 to 25° C.

Cleaning is a three-fold stepwise washing with purified water onsemitransparent cassette (with lower limit of particles cutoff rangingfrom 1 to 30 kDa) upon finishing oxidation, alkylation andhydrazinolysis, as well as conjugation steps.Conjugation is carried out with the use of water-soluble copolymer ofgeneral formula (I) comprising in its chain from 3 to-20% hydrazidegroups.

In the case of carbodiimide method of conjugation poly-1,4-ethylenepiperazin is first alkylated and then subjected to oxidation in aqueousenvironment. In this process alkylation is performed by using haloalkaneacid.

In carbodiimide method cleaning is done in a stepwise manner by washingwith purified water on semitransparent cassettes (with lower limit ofparticles cutoff ranging from 1 to 30 kDa) upon finishing the steps ofcopolymer obtaining and conjugation.The conjugation is carried out with the use of water-soluble copolymerof N-oxide poly-1,4-ethylene piperazin of general formula (I) comprisingin its chain up to 25% of carboxyl groups, and use is made of anywater-soluble carbodiimides in amount from 3 to 50% from the copolymerinvolved in the reaction.

The invention subject matter is also an active conjugate ingredientpossessing the properties of inhibiting hyperplasia of connective tissueand anti-inflammatory action, and being produced by any claimed methodsfor preparation of a medicinal agent in a form of administrationselected from a suppository, ointment, injection, or cosmetic cream,including drugs for veterinary.

Given below is the detailed description of the claimed method with theuse of azide and carbodiimide methods of conjugation of production ofLongidaza having particular properties described in detail in 10Examples and presented in Table 1.

Under conditions of claimed technology of polymer carrier andhyaluronidase conjugates production, as it was found, there occurs thedesired distribution of functional units in the copolymer structure,wherein polymer carrier and conjugates on its base become resistant inthe process of long-term storage. Said result is achieved by performingthe reaction of polyethylene piperazin oxidation under dissociatingconditions by way of adding urea. At that, non-covalent intermolecularbonds of polyethylene piperazin get destroyed, the reactions involveseparate polymer molecules, rather than molecules in the composition ofa micelle, the desired uniform distribution of functional groups alongthe polymer chain is achieved, there get increased enzyme activity (by30 to 65%, see Table 1), the conjugation degree and target product yield(by 15 to 20%, see Table 1), and stability of hyaluronidase conjugate.Enzyme activity of Longidaza preparations kept in storage at atemperature of 2 to 8° C. within 1 year dropped by 4 to 6% for thespecimens obtained with the use of urea, and by 24 to-27% without use ofurea (Table 1, Example 4 and Example 9).

The conjugate is produced by the following two methods:the method for hyaluronidase conjugate production using the azide methodof conjugation; andthe method for hyaluronidase conjugate production using the carbodiimidemethod of conjugation.Poly-1,4-ethylene piperazin (PEP) with molecular weight from 20 to 60kDa is used as a feedstock for all methods of production.PEP results from cationic polymerization, which is an efficient methodof synthesis of monodisperse high-molecular compounds having presetmolecular weight and structure,

1. Method for Production of Conjugate Using Azide Method of Conjugation.

This production method consists of the basic steps of the process,namely, oxidation in the presence of urea, alkylation, hydrazinolysis,conduct of conjugation using azide method,

Oxidation of poly-1,4-ethylene piperazin is carried out in an acidicmedium by means of hydrogen peroxide in the presence of urea at atemperature of 45 to 55° C. for 12 to 24 hours in a water-jacketedreaction vessel. Then, the reaction feed is diluted, filtered in acartridge filter provided with a filtering element having a pore-size of0.45 nm, undergoes diafiltration in an ultrafiltration unit withcassettes having the lower limit of particles cutoff of 5 kDa, and getsfiltered in a cartridge filter provided with a filtering element havinga pore-size of 0.22 nM. Thereafter, the resultant product is dried, andtested for compliance with the regulatory document requirements and turnin for storage.

At the second step N-oxide poly-1,4-ethylene piperazin undergoesalkylation by means of ether of haloalkane acid in a water-organicsolution (a mixture of water and N-methyl formamide) with agitationwithin a temperature range of 35 to 50° C. for 4 to 6 hours, and then,hydrazinolysis of alkyl derivative of polyethylene piperazin is done byprocessing using hydrazine hydrate at temperatures of 2 to 8° C. Thereaction feed is filtered in a cartridge filter provided with afiltering element having a pore-size of 0.45 nm, then it undergoescleaning in an ultrafiltration unit with cassettes having the lowerlimit of particles cutoff of 10 kDa (percentage of hydrazine hydrate notexceeding 0.001%), thereafter the feed is subjected to sterilizingfiltration in a cartridge filter and reconstituted in glass vials.Finally, it is tested for compliance with the requirements of theregulatory document and turned in for storage.

At the last third step the process of conjugation of hyaluronidase withhydrazide compound of carboxymethyl-containing N-oxide polyethylenepiperazin comprising in its chain more than 3% of hydrazide groups isperformed using azide method by treatment with sodium nitrite at pH=0-1and subsequent conjugation of azide derivate with hyaluronidase for 18to 22 hours at indoor temperature. The hyaluronidase conjugate isfiltered, cleaned by washing in an ultrafiltration unit (cassettes withthe lower limit of particles cutoff of 5 kDa), filter-sterilized andspray-freeze dried (lyophilized). Test results are shown in Table 1(Examples 1-3).

The claimed production method, which includes the steps of alkylation,oxidation (but without urea addition), hydrazinolysis, conjugation byazide method and cleaning, is used to obtain a Longidaza specimen. Theresults of this specimen testing are presented in Table 1 (Example 4).The comparative studies demonstrate that addition of urea increasesenzyme activity, degree of conjugation and yield of target product, andconjugate stability (Table 1).

The derived freeze-dried (lyophilized) product is reconstituted in glassvials and turned in for storage in the form of an active conjugateingredient having the activity similar to that of Longidaza®.

Further on, the obtained active pharmaceutical ingredient is used forproduction of liquid or freeze-dried injectable dosage form. In thisregard the solution is diluted to ensure that in 1 ml of solution a theenzyme activity is 3,000 IU (therapeutic dose), and it is tested in allparameters for compliance with the requirements of the regulatorydocument for finished-dosage form. After that the solution can befreeze-dried (lyophilized medicinal preparation) or, without drying, theliquid medicinal preparation get packaged, labeled and tested in allparameters for compliance with the requirements set forth in theregulatory document for finished-dosage form.

The carbodiimide method, claimed in the present invention, makespossible the process performance without use of organic solvents andharmful, the end product, i.e., the hyaluronidase conjugate withderivatives of polyethylene piperazin, improvement and stability.

2. Method of Longidaza Production with the Use of Carbodiimide Method ofConjugation.

The sequential production process consists of the basic steps, namely,alkylation, oxidation in the presence of urea, conduct of conjugationusing carbodiimide method.

There is used a poly-1,4-ethylene piperazin with preset properties (seeabove), which is alkylated by bromo-acetic acid in aqueous environmentat a temperature of 40 to 95° C. and weight ratio (85:15)-(70:30),respectively. The feed is mixed at a temperature of 50 to 70° C. for 3hours, then urea, acetic acid and nitrogen peroxide are added to thereaction mass, and thereafter the resultant mixture is kept, beingcontinuously stirred, for 18 to 22 hours. Then, the oxidation processcompleteness is checked with the aid of ammonia test (similar to Example1). The reaction mass is filtered in a cartridge filter provided with afiltering element having a pore-size of 0.45 nm. The polymer is cleanedby washing in an ultrafiltration unit with the lower limit of particlescutoff of 5 kDa to a nitrogen peroxide content of not more than 0.001%(the nitrogen peroxide content test). The solution is concentrated to10-15%, the content of carboxyl groups in the polymer shall not be lessthan 6%. The calculated amount of hyaluronidase is introduced into thereaction medium, while stirring, and the medium pH is brought up to avalue of 4.8.

With stirring being continued, a water-soluble carbodiimide is added toensure that its content is 25 to 50% of added hyaluronidase (expressedas protein). The reaction feed is kept at a temperature of 0 to 25° C.for 1 to 1.5 hours, and then there is checked the degree of conjugationwhich shall be not less than 95%. In case of a positive result thereaction feed is washed with purified water, alkalified to pH=6.8 to 7.0and filtered in disc filter equipped with AP-15 deep-bed filter. Afterprefiltration the reaction feed cleaning is continuing by washing withpurified water on a Pellicon-cassette to ensure that the total amount ofwater would be at least 200 liters per 1 kg of dry matter. Then, thesolution ingredient having the Longidaza® activity is concentrated,sterile filtered in a cartridge filter fitted with a filtering membranewith a pore-size of 0.22 nm and reconstituted in glass vials in the formof a liquid ingredient. The resultant sterile masterbatch can also befreeze-dried sterile with obtaining in this case an ingredient havingthe activity similar to that of Longidaza® preparation in dry state.Thereafter it is checked for compliance with the requirements of theregulatory document and turned in for storage as an end product. Theingredient test results are given in Table 1 (Examples 6 to 8).

The claimed production method, which includes the steps of alkylation,oxidation, but without urea addition, conjugation by carbodiimide methodand cleaning procedure, was used to get the Longidaza specimen, the testresults of which are presented in Table 1 (Example 9). In the same wayas for the case of the Longidaza production with the use of azide methodof conjugation, urea addition increases enzyme activity, conjugationdegree and target product yield, and the conjugate stability (Table 1,Table 2).

Sterile solution of the hyaluronidase conjugate is used for the intendedpurpose. The resultant active pharmaceutical ingredient is used forpreparation of liquid or freeze-dried injectable dosage forms. With thisin view this solution is diluted to ensure that in 1 ml of solution theenzyme activity is 3,000 IU (therapeutic dose) and reconstituted inglass vials and freeze-dried (lyophilized medicinal preparation) or,without drying, the liquid medicinal preparation get packaged, labeledand tested in all parameters for compliance with the requirements setforth in the regulatory document for finished-dosage form.

In the process of hyaluronidase conjugate with polyethylene piperazinderivatives the ultrafiltration cleaning on cassettes with particlescutoff of 1 to 30 kDa is used to remove process impurities. Washing isdone on completion of alkylation and oxidation steps, and at the end ofthe production process. The claimed carbodiimide method of conjugationis carried out exclusively in aqueous environment.

Thus, the essence of the claimed method consists in a new approach inthe technology of production of immobilized compounds on a polymercarrier of new class in the form of water-soluble PEP derivatives as perazide and carbodiimide methods. Copolymer is obtained by chemicalmodification from PEP in the presence of urea, which in the process ofconjugation significantly boosts the hyaluronidase activity, degree ofconjugation, yield of preparation having the Longidaza activity and itsstability in storage.

Analysis of results of production of hyaluronidase conjugate withderivatives of polyethylene piperazin are presented in Table 1 (Examples1 thru 10) indicates high yield of the target product. At that, theprocess with the use of carbodiimide method provides for higher degreeof conjugation and end product activity, and the process is carried outexclusively in aqueous environment.The inventive step of developed methods of production of hyaluronidaseconjugate with derivatives of polyethylene piperazin is verified byabsence in this business profile of continuous processes, and by thefact that production of N-oxide polyethylene piperazin was carried outwith the use of urea. In industrial setting the carbodiimide method ismore preferable than the azide method. The employment of developedmethods in production of long-acting forms of compounds of variousapplications improves the production of a new preparation having theactivity higher than the activity of Longidaza® preparation.

The studies of decomposition of copolymers of N-oxide 1,4-ethylenepiperazin, (N-carboxymethyl)-1,4-ethylene piperazin and 1,4-ethylenepiperazin have shown that at a temperature not above 70° C. in aqueousenvironment with pH not exceeding 9, decomposition occurs exclusivelyover N-oxide units. Thermal decomposition of N-oxides proceeds by way ofMeisenheimer rearrangement.

Copolymer having uniform distribution of various units should not havebig portions consisting of units of only one type. Thus, with N-oxideunits content of 50% and more and their uniform distribution along thepolymer chain, the copolymer shall not have big portions composed ofonly unsubstituted units of 1,4-ethylene piperazin (units 1). In lightof this, there has been developed a method of qualitative andquantitative evaluation of uniformity of distribution of unitscomprising N-oxide groups along the copolymer chain. The method consistsin the copolymer controlled decomposition with subsequentphysic-chemical analysis of resultant fragments. It was found that withurea use in the process of oxidation the decomposition products do notcontain low-molecular polymer comprising one-type units:

where l=3 and above.

Availability of this substance in the copolymer decomposition productsis indicative of significant non-uniformity of N-oxide unitsdistribution in starting copolymer.

When analyzing copolymer of series 111114 using the degradation methodthe following substance was extracted

Upon completion of quantitative and qualitative analysis of compositionof this substance one can judge on the qualitative and quantitativeprofile of N-oxide groups distribution in the copolymer under study.However, the developed measuring procedure is time consuming (themonitored degradation of N-oxide groups of a copolymer takes 5 weeks)and big amount of the substance to be analyzed. With this in view, therewas designed a qualitative method for rapid assessment of unitsdistribution uniformity.

The analysis of various specimens of copolymers using the DSC(Differential Scanning Calorimetry) method it was demonstrated that thepolymers of this type vigorously degrade at a temperature of about 160°C. with heat liberation. At this, various forms of peak exotherm ofdecomposition of specimens produced with or without urea added. Thus,there were analyzed two specimens of a polymer carrier having similarquantitative parameters, namely, molecular weight, molecular-weightdistribution, number of carboxyl and N-oxide groups produced with theuse of urea at the oxidation step and without urea addition. In thiscase, the conjugates, obtained on one polymer carrier, were stable, andthose obtained on another carrier were not stable.

The DSC curves for polymers are shown in FIGS. 1 and 2.The DSC curves (FIGS. 1 and 2) demonstrate significant difference ofratio of peak intensities within a temperature range of 150 to 170° C.The obtained results indicate the relation between the polymer structure(units distribution profile) and its thermal stability. The receivedresults were verified by analysis of other carriers, and theydemonstrate a possibility of distinguishing the specimens of polymercarriers by different stability due to functional groups arrangement inmolecules of a polymer carrier.

Given below are the particular parameters accumulated in Table 1 andpresented in Examples 1-3 and 6-8. Table 1 also presents the results ofwholesale scale series (Example 5 and Example 10). Examples 4 and 9contain the data verifying the advantages of the method of production inthe presence of urea.

EXAMPLE 1

The method of production of hyaluronidase conjugate with derivatives ofpolyethylene piperazin using azide method of conjugation.Taken are 200 grams of, preliminary chopped and cleaned by a method ofquadruple washing with purified water, chips of poly-1,4-ethylenepiperazin with a molecular weight of 50 kDa. Chips are oxidized in amixture of 50 ml of acetic acid, 140 ml of 30-% solution of hydrogenperoxide in water, in the presence of 50 grams of urea. Water is addedin such a way that the PEP content is 15 wt. %, i.e. the volume ofreaction medium should be 1.5 liters. The reaction feed is heated up to50° C. and, being continuously stirred, is kept for 24 hours. Theoxidation completeness is checked with the aid of the ammonia testing.This testing consists in the following: 10 ml of 25-% solution ofammonia are added to 1 ml of reaction feed. Absence of turbidity insolution, after keeping it for 20 minutes, is considered as a positiveresult. In case of the positive result the reaction feed is diluted,filtered in a cartridge filter fit with a filtering element with apore-size of 0.45 nm, cleaned and concentrated in an ultrafiltrationunit with cassettes having the lower limit of particles cutoff of 5 kDa,and filtered in a cartridge filter equipped with a filtering elementwith a pore-size of 0.22 nm. Thereafter, it is freeze-dried, and 204grams of N-oxide poly-1,4-ethylene piperazin are obtained in the form oflyophilizate, which is checked for compliance with the requirements ofthe regulatory document and turned in for storage.

The resultant N-oxide poly-1,4-ethylene piperazin is dissolved in 1,500ml of distilled water, and 6,000 ml of N-methyl formamide is added. Theresultant solution is added with 275 ml of bromalkane ether and, beingcontinuously stirred, is allowed to stand at temperatures of 35-50° C.for 4 to 6 hours. Then the reaction mass is cooled down to 2-8° C. and,being stirred, added with 250 ml of hydrazine hydrate. The reaction feedis diluted 10 times with purified water, filtered in a cartridge filterfit with a filtering element with a pore-size of 0.45 nm, and cleaned bywashing on Pellicon-cassettes (with the lower limit of particles cutoffof 10 kDa) to a content of trace amount of hydrazine hydrate (not morethan 0.001%). Further the reaction feed is concentrated to 10-12% (asper target substance) sterile filtered and reconstituted in glass vials.There are obtained 2,200 ml of solution of hydrazide derivative ofN-oxide poly-1,4-ethylene piperazin, which is analyzed for compliancewith the requirements of the regulatory document, and turned in forstorage.

The solution obtained during the previous step is cooled down to 2 to 6°C. and, being stirred and cooled, it is added with hydrochloric acid topH=0-1. Then, with stirring and cooling continued, it is added batchwisewith 360 grams of sodium nitrite and the reaction mass is allowed tostand for 1.5 hours. After that the reaction medium is rendered alkalineby moist-free ash to pH=6.8-7 and, at a temperature not exceeding 10°C., while stirring 10-% solution, containing 65 grams of hyaluronidasepreparation (70% of protein), is added. The reaction feed remainsstirred for 1.5 hours, then, a sample is taken to check the conjugationdegree. In the case of positive result of the conjugation degreechecking (not less than 70%) the reaction mass is filtered in a discfilter fit with a deep-bed filter, AP-15 type, and washed in 50 litersof purified water in an ultrafiltration unit with cassettes having thelower limit of particles cutoff of 5 kDa. The solution is concentrated,sterile filtered and filled in sterile glass vials (liquid ingredient).Sterile solution is also freeze-dried and packed in sterile glass vials(freeze-dried ingredient). There are obtained 231 grams of ingredient,86-% yield. The results of testing the obtained conjugate ingredient arepresented in B Table 1 (Example 1). The resultant ingredient is used forpreparation a finished medicinal product with a type of activity beingidentical to that of known “Longidaza preparation in the form of vaginaland rectal suppository of 3,000 IU”.

EXAMPLE 2

The procedure is similar to that of Example 1, with the difference thatthere is used chopped and purified PEP in amount of 200 g with amolecular weight of 40 kDa, and that 60 grams of urea are used at theoxidation step. There are obtained 227 grams of ingredient with a typeof activity being identical to the activity of known Longidazapreparation in the form of lyophilizate, 84-% yield. The analysisresults are presented in Table 1, Example 2.The resultant ingredient is used for preparation of cream and ointmentsfor external use, 1000 IU.

EXAMPLE 3

The procedure is similar to that of Example 1, with the difference thatthere is used chopped and purified PEP in amount of 200 g with amolecular weight of 55 kDa, and that 80 grams of urea are used at theoxidation step. There are obtained 235 grams of ingredient with a typeof activity being identical to the activity of known Longidazapreparation in the form of lyophilizate, 87-% yield. The analysisresults are presented in Table 1, Example 3.This Ingredient is used for preparation of injectable dosage form.

EXAMPLE 4

The procedure is similar to that of Example 1, with the difference thaturea is not added at the oxidation step. The resultant ingredientamounting to 215 grams has the activity of Longidaza® in the form oflyophilizate, 74% yield. The results of Longidaza testing are given inTable 1, Example 4.The ingredient having the activity being identical with the activity ofknown drug “Longidaza” is used for preparation of injectable dosageform.

EXAMPLE 5

The procedure is similar to that of Example 1, with the difference thatthere is used chopped and purified PEP in amount of 4,000 g with amolecular weight of 45 kDa, and that 2,000 grams of urea are used at theoxidation step. There are obtained 4,700 grams of ingredient with a typeof activity identical to the activity of known Longidaza® preparation inthe form of lyophilizate, 87-% yield. The test results are presented inTable 1 (Example 5).Batching up the required amount of produced ingredient in suppository,ointment or cream bases we get respectively suppositories, creams,ointments comprising active ingredient. It is also used for preparationof injectable dosage form.

EXAMPLE 6

The method with the use of carbodiimide method of conjugation.Poly-1,4-ethylene piperazin in amount of 200 g with a molecular mass of30 kDa is added to 1.2 liters of boiling-water solution comprising 58grams of bromo-acetic acid, and keep heating it at a temperature of 70°C. for 3 hours, continuously stirring said solution. Then, the reactionmass is added with 40 ml of acetic acid, 140 ml of 30-% water solution aof nitrogen peroxide, 50 grams of urea and all are stirred at atemperature of 38-42° C. for 18 to 20 hours. Further, the oxidationstate completeness is checked with the aid of ammonia test (this test isdescribed above). In the case of a positive result the reaction feed isdiluted to a concentration of 1 to 2%, and filtered in a cartridgefilter fit with a filtering element having a pore-size of 0.45 nm,cleaned by washing by purified water on a Pellicon-cassette having thelower limit of particles cutoff equal to 5 kDa. In the process ofwashing the solution is rendered alkaline to a value of pH=11.0-11.5with the purpose of complete cleaning to remove traces of low molecularorganic acids. The solution cleaning is continued until the nitrogenperoxide content becomes not more than 0.001% (nitrogen peroxide test),and the solution is concentrated to 10% of the target substance. Theresultant solution is acidified by hydrochloric acid to pH=4.8-4.9,after that 50 grams of hyaluronidase are added. The pH value issubjected to adjustment; it should be 4.8 to 4.9. The reaction feed,being stirred, is added with a solution comprising 2.1 grams ofN-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride in 210 mlof water for 1.5 to 2 hours. The conjugation reaction is carried out ata temperature of 2 to 25° C. After adding the last portion of condensingagent solution the reaction mass is allowed to stand<being stirred, for30 minutes at a preset temperature, and a sample is taken to determinethe conjugation degree. In the case of a positive result conjugationdegree should be at least 90%, the reaction mass is allowed to stand for18 to 20 hours, it is rendered alkaline by means of moist-free ash to avalue of pH=6.8 to 7.0, washed and filtered in a disc filter with afiltering material in the form of a deep-bed filter, AP-15 type. Then,the reaction mass is cleaned by purified water in an ultrafiltrationunit on a Pellicon-cassette with the lower limit of particles cutoffequal to 5 kDa. The cleaned solution is concentrated to a content of8-10%, and sterile filtered in a cartridge filter fit with a filteringelement having a pore-size of 0.22 nm. The resultant solution ofhyaluronidase conjugate with derivatives of polyethylene piperazin canbe diluted to a concentration containing in one milliliter of solution3,000 IU of enzyme activity, and finally get an injectable dosage form(in liquid form) or freeze-dried (in lyophilized form). In the case offreeze-drying, 261 grams of ingredient are obtained with a type of itsactivity being identical to the activity of known Longidaza®preparation, 97-% yield. The test results are presented in Table 1,Example 6.Batching up the required amount of produced ingredient in suppositorybase we get respectively suppositories.

EXAMPLE 7

The procedure is similar to that of Example 6, with the difference thatthere is used chopped and purified PEP in amount of 200 g with amolecular weight of 38 kDa, and that 60 grams of urea are used at theoxidation step. There are obtained 265 grams of ingredient with a typeof activity identical to the activity of known Longidaza® preparation inthe form of lyophilizate, 98-% yield. Test results are presented inTable 1, Example 7.This ingredient is used for preparation of an ointment.

EXAMPLE 8

The procedure is similar to that of Example 6, with the difference thatthere is used chopped and purified PEP in amount of 200 g with amolecular weight of 26 kDa, and that 70 grams of urea are used at theoxidation step. There are obtained 264 grams of ingredient with a typeof activity identical to the activity of known Longidaza® preparation inthe form of lyophilizate, 98-% yield. Test results are presented inTable 1, Example 8.This ingredient is used for preparation of injectable dosage form.

EXAMPLE 9

The procedure is similar to that of Example 6, with the difference thatthere is used chopped and purified PEP in amount of 200 g with amolecular weight of 26 kDa, and that at the oxidation step no urea isadded. There are obtained 217 grams of the ingredient with a type ofactivity identical to that one of the known Longidaza preparation in theform of lyophilizate, 82-% yield. The test results are presented inTable 1, Example 9.The ingredient is used for preparation of injectable dosage formcomprising Longidaza.

EXAMPLE 10

The procedure is similar to that of Example 6, with the difference thatthere is used chopped and purified PEP in amount of 4,000 g with amolecular weight of 36 kDa, and that at the oxidation step 1,500 gramsof urea is added. There are obtained 5,130 grams of the ingredient withthe type of activity being identical to that of known Longidazapreparation in the form of lyophilizate, 95-% yield. The test resultsare presented in Table 1 (Example 10).Batching up the required amount of produced ingredient in suppository,ointment or cream bases we get respectively suppositories, creams,ointments comprising active ingredient. It is also used for preparationof injectable dosage form.The claimed continuous carbodiimide method makes it possible to excludefrom the process organic solvents, to simplify the process, upgrade thequality and increase the target product yield, to achieve high value ofthe conjugation degree, increase the stability of Longidaza® (infreeze-dried and liquid form) under its exposure to variousenvironmental effect in the process of its processing and storage.

The claimed methods provide for improvement of processes of productionof long-acting conjugates based on the new class carriers of compoundsin the form of water-soluble derivatives of heterocyclic aliphaticaminopolymer N-oxides, comprising in their chain azide or carboxylgroups, and non-stable compounds of protein or other nature comprisingactive amine groups in its composition. The method has great practicalimportance in providing for the pharmaceutical production long-actingmedicinal preparations being in great demand at the market of medicinalpreparations, and it can also be used for preparation of veterinarymedicines. In perspective, the developed production processes can bewidely used for solving great many persistent problems in varioussectors of national economy of the country as a whole.

TABLE 1 Results of analytical monitoring of series of hyaluronidaseconjugate with copolymer of N-oxide 1,4-ethylene piperazin and(N-carboxymethyl)-1,4-ethylene piperazin halogenide derived in EXAMPLES1 through 10. Enzyme activity Enzyme activity after Enzyme activityTarget Production of medicinal Method of at the time of production, 1year of storage*⁾, decrease within 1 Conjugation product preparationwith production IU/mg of drug IU/mg of drug year, % degree, % yield, %Longidaza EXAMPLE 1 221 212 4 85 86 Suppositories EXAMPLE 2 230 219 5 8484 Cream, ointment EXAMPLE 3 242 230 5 86 87 Injections EXAMPLE 4 168123 27 65 74 Injections w/o urea EXAMPLE 5 235 221 6 85 87Suppositories, injections, industrial cream, ointment batch 4, kgEXAMPLE 6 265 254 4 96 97 Suppositories EXAMPLE 7 280 266 5 99 98 CreamEXAMPLE 8 275 264 4 99 98 Injections EXAMPLE 9 175 133 24 85 82Injections w/o urea EXAMPLE 10 289 272 6 97 95 Suppositories,injections, industrial cream, ointment batch 4, kg *⁾Storage at t = 2 to8° C.

TABLE 2 Study of stability during long-term storage of Longidazaproduced with urea use (EXAMPLE 3 

 EXAMPLE 8) and without urea use (EXAMPLE 9) as per ‘cell-free protein’(Protein S) parameter Cell-free protein, % Method of at the time of 3 69 12 production production months months months months EXAMPLE 3 14 1516 16 17 EXAMPLE 8 1 1 2 2 3 EXAMPLE 9 15 26 30 32 33 (w/o urea)

1. A method for preparation of active conjugate of hyaluronidase enzymewith copolymer comprising N-oxide 1,4-ethylene piperazine and(N-carboxymethyl)-1,4-ethylene piperazine halogenide with the use ofcarbodiimide or azide method of conjugation, cleaning and spray-freezedrying characterized in that conjugation is made with the use ofwater-soluble copolymer, which is a copolymer of N-oxidea 1,4-ethylenepiperazine, (N-carboxymethyl)-1,4-ethylene piperazine or its hydrazide,and 1,4-ethylene piperazine of general formula

where n is ranging from 40% to 90% of total number of units; m isranging from 3 to 40% of total number of units; n+m+1=100%, obtainedfrom poly-1,4-ethylene piperazine by way of oxidation, alkylation, andin the case of azide method of hydrazinolysis, wherein the oxidation isperformed by means of oxidizing agent being able to generate oxygenunder normal conditions in presence of urea, and alkylation is performedby means of lower haloalkane acid or its alkyl ether.
 2. Method of claim1 characterized in that the oxidation step is carried out with additionof 1 to 10%, preferably 3 to 6 wt. % of urea per total mass of reactionfeed, including water.
 3. Method of claim 1 characterized in that theconjugation is carried out with the use of hyaluronidase taken fromseminal glands of animals.
 4. Method of claim 1 characterized in thatstep-by-step cleaning is performed by washing using purified water onsemitransparent cassettes with lower limit of particles cutoff rangingfrom 1 to 30 kDa.
 5. A method for preparation of active conjugate ofhyaluronidase enzyme with a copolymer comprising N-oxide 1,4-ethylenepiperazine and (N-carboxymethyl)-1,4-ethylene piperazine halogenide withthe use of azide method of conjugation, cleaning, concentrating anddiluting or spray-freeze drying characterized in that conjugation ismade with the use of water-soluble copolymer, which is a copolymerN-oxidea 1,4-ethylene piperazine, hydrazide of(N-carboxymethyl)-1,4-ethylene piperazine and 1,4-ethylene piperazine ofgeneral formula

where n is ranging from 40% to 90% of total number of units; m isranging from 3 to 20% of total number of units; n+m+1=100%, obtainedfrom poly-1,4-ethylene piperazine by way of oxidation, alkylation, andin the case of azide method of hydrazinolysis, wherein the oxidation isperformed by means of oxidizing agent being able to generate oxygenunder normal conditions in presence of urea, alkylation is carried outby alkyl ether of haloalkane acid, wherein the alkylation state andhydrazinolysis are combined.
 6. Method of claim 5 characterized in thatthe oxidation state is carried out with addition of 1 to 10%, preferably3 to 6 wt % of urea per total mass of reaction feed, including water. 7.Method of claim 5 characterized in that cleaning is a three-foldstepwise washing with purified water on semitransparent cassettes (withlower limit of particles cutoff ranging from 1 to 30 kDa) upon finishingoxidation, alkylation and hydrazinolysis, as well as conjugation steps.8. Method of claim 5 characterized in that the conjugation is made withthe use of water-soluble copolymer of N-oxidea

-1,4-ethylene piperazine comprising in its chain from 3 to 20% ofhydrazide groups
 9. Method of claim 5 characterized in that theconjugation with hyaluronidase is carried out using azide method attemperatures ranging from 0 to 25° C.
 10. A method for preparation ofactive conjugate of hyaluronidase enzyme with copolymer comprisingN-oxide 1,4-ethylene piperazine and (N-carboxymethyl)-1,4-ethylenepiperazine halogenide with the use of carbodiimide method ofconjugation, cleaning, diluting and concentrating or spray-freeze dryingcharacterized in that the conjugation is done with the use ofwater-soluble copolymer, which is a copolymer N-oxidea 1,4-ethylenepiperazine, (N-carboxymethyl)-1,4-ethylene piperazine and 1,4-ethylenepiperazine of general formula

where n is ranging from 40% to 90% of total number of units; m isranging from 3 to 40% of total number of units; n+m+1=100%, obtainedfrom poly-1,4-ethylene piperazine by alkylation and oxidation in aqueousenvironment, wherein oxidation is carried out by means of oxydizingagent, being able to generate atomical oxygen under normal conditions inpresence of urea, alkylation is carried out by using haloalkane acid.11. Method of claim 10 characterized in that the oxidation step iscarried out with addition of 1 to 10%, preferably 3 to 6% wt %, of ureaper total mass of reaction feed, including water.
 12. Method of claim10, characterized in that cleaning is carried out step-by-step usingpurified water on semitransparent cassettes (with lower limit ofparticles cutoff ranging from 1 to 30 kDa) upon finishing the steps ofcopolymer obtaining and conjugation.
 13. Method of claim 10,characterized in that the process is carried out with the use ofwater-soluble copolymers of N-oxidea

-1,4-ethylene piperazine comprising in their chain up to 25% of carboxylgroups.
 14. Method of production as described in claim 10, characterizedin that the conjugation step is carried out with the use of anywater-soluble carbodiimides amounting from 3 to 50 wt % of proteinproduced in the course of reaction.
 15. A medicinal preparation,possessing the properties of simultaneously inhibiting connective tissuehyperplasia and anti-inflammatory action, produced using this method asdescribed in any of claims 1 through 14 in the form of a suppository,liniment, injection or cosmetic cream.